Faster measurement of viable particles in UPW

by Robert P. Donovan

Culturing methods1, the gold standard for measuring bacteria in ultrapure water (UPW), take 24 to 48 hours or more to report a result. When you need faster response times, here are some options:

Epifluorescence2: Staining of bacteria collected on a filter so that they fluoresce under appropriate excitation and can be enumerated under a fluorescent microscope (one to two hours).

Carboxy fluorescein diacetate (CFDA)3, 4: Staining of bacteria on a filter with CFDA, which reacts with esterase from a living cell to form a luminescent, countable fluorescein (one to two hours).

Adenosine triphosphate (ATP; C10H16N5O13P3)5: Bacteria contains ATP which, when released, reacts with luciferin (a chemical present in bioluminescent organisms such as fireflies) and oxygen in the presence of the enzyme, luciferase, to form a luminescent product within a short period. This type of bioluminescent interaction, utilizing the antibody lysin as an ATP releasing agent, has recently been touted as both a detector and antidote for anthrax.6, 7 Particles collected from air on a filter are treated with the appropriate chemicals rather than culturing media. While the method development assumes an aerosol sample, the principles also apply to water sampling.5

Polymersase chain reaction (PCR)8: DNA splitting and replicating to magnify the DNA concentration and simplify counting by electrophoresis or other appropriate methods (six-hour response time between sampling and analytic results).

While faster than the standard culturing method, none of these methods are real-time or near-real-time. However, a few near-real-time detectors are in development, most based on either intrinsic fluorescence—no chemical conditioning required—or light scattering.

One example of the latter is Micro Imaging Technology's (Laguna Beach, CA) MIT system, which uses light scattering to characterize waterborne particles in near real time. This design (see “Rapid contamination detection technology patent granted,” CleanRooms August 2002, page 1) is an extension of conventional optical particle counters, which collect an integrated scattered light signal at a fixed scattering angle.

The MIT system collects differential scattered light at a large number of angles in a spherical space surrounding the light scattering particles. The signature of a scattering particle detected by this system can then be compared with those in a library of signatures previously assembled from known species to identify the composition of a specific particle.

Classification of an aerosol particle as viable or non-viable can be made in milliseconds, and often so can the composition of a specific viable aerosol particle. A patent for this approach was granted earlier this year but the product is still in the prototype stage.

Recent concern over biological weapons has spawned an intense effort to develop faster and more sensitive biodetectors, many based on intrinsic fluorescence of aerosol samples.9

Similar technologies can often be adapted to water sampling and detection so that prospects for more rapid methods of counting viable particles in UPW are now more promising than ever. Next month's column will describe one commercially available instrument that uses intrinsic fluorescence to detect bioaerosols in near real time.

Robert P. Donovan is a process engineer assigned to the Sandia National Laboratories and a monthly columnist for CleanRooms magazine. He can be reached at [email protected].


  • 1. American Society of Test and Measurement (ASTM) F 1094-87 (Reapproved 1999), Standard Test Methods for Microbiological Monitoring of Water Used for Processing Electron and Microelectronic Devices by Direct Pressure Tap Sampling Valve and by the Presterilized Plastic Bag Method R (1999) (ASTM, 100 Barr Harbor Dr., West Conshohocken, PA 19428).
  • 2. Mittelman, M. E., P. W. Johnson and J. McN. Sieburth, “Epifluorescence Microscopy, a Rapid Method for Enumerating Viable and Nonviable Bacteria in Ultrapure Water Systems,” Microcontamination Vol 1, No. 2, 1983, pp. 32-37, 52.
  • 3. Osawa, M. et al., “Analysis of Bacteria in Ultrapure Water Systems Using Fluorescent Probe and Culture Method,” 1998 SPWCC Proceedings, pp. 121-139 (Balazs Analytical Laboratory, 252 Humboldt Court, Sunnyvale, CA 94089-1315).
  • 4. Stancyzk, B., S. Tan and D. Jones, “Rapid Detection of Viable Bacteria in High-Purity Water in the Microelectronics Industry,” presentation at Semiconductor Water Treatment Executive Forum, WATERTECH—Portland, OR, December 2001 (Ultrapure Water, POB 621669, Littleton, CO 80162-1669).
  • 5. Manabe, T., “ATP Monitor,” Ultraclean Technology Handbook, Vol 1, Ultrapure Water, T. Ohmi, editor, Marcel Dekker, Inc., New York, NY, 1993, pp. 595-600.
  • 6. Wade, N., “Anthrax Study May Yield Remedy,” The New York Times, 08/22/02 [http://www.nytimes. com/2002/08/22/science/22ANTH.html].
  • 7. Rosovitz, M. J. and S. H. Leppia, “Virus Deals Anthrax a Killer Blow,” Nature 418, 22 August 2002, pp 825-826.
  • 8. Pepper, I. L. et. al., “A Rapid and Systematic Analytical Method for Measuring Bacterial Contaminants in Ultrapure Water,” 1993 SPWCC Proceedings, pp. 50-62 (Balazs Analytical Laboratory, 252 Humboldt Court, Sunnyvale, CA 94089-1315).
  • 9. National Institute of Justice, “An Introduction to Biological Agent Detection Equipment for Emergency First Responders,” NIJ Guide 101-00, December 2001.


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